Chyong Jy Nien

Nonlinear Optical Macroscopic Assessment of 3-D Corneal Collagen Organization and Axial Biomechanics

PURPOSE To characterize and quantify the collagen fiber (lamellar) organization of human corneas in three dimensions by using nonlinear optical high-resolution macroscopy (NLO-HRMac) and to correlate these findings with mechanical data obtained by indentation testing of corneal flaps. METHODS Twelve corneas from 10 donors were studied. Vibratome sections, 200 μm thick, from five donor eyes were cut along the vertical meridian from limbus to limbus (arc length, 12 mm). Backscattered second harmonic-generated (SHG) NLO signals from these sections were collected as a series of overlapping 3-D images, which were concatenated to form a single 3-D mosaic (pixel resolution: 0.44 μm lateral, 2 μm axial). Collagen fiber intertwining was quantified by determining branching point density as a function of stromal depth. Mechanical testing was performed on corneal flaps from seven additional eyes. Corneas were cut into three layers (anterior, middle, and posterior) using a femtosecond surgical laser system and underwent indentation testing to determine the elastic modulus for each layer. RESULTS The 3-D reconstructions revealed complex collagen fiber branching patterns in the anterior cornea, with fibers extending from the anterior limiting lamina (ALL, Bowmans layer), intertwining with deeper fibers and reinserting back to the ALL, forming bow spring-like structures. Measured branching-point density was four times higher in the anterior third of the cornea than in the posterior third and decreased logarithmically with increasing distance from the ALL. Indentation testing showed an eightfold increase in elastic modulus in the anterior stroma. CONCLUSIONS The axial gradient in lamellar intertwining appears to be associated with an axial gradient in the effective elastic modulus of the cornea, suggesting that collagen fiber intertwining and formation of bow spring-like structures provide structural support similar to cross-beams in bridges and large-scale structures. Future studies are necessary to determine the role of radial and axial structural-mechanical heterogeneity in controlling corneal shape and in the development of keratoconus, astigmatism, and other refractive errors.

Effects of age and dysfunction on human meibomian glands.

OBJECTIVE To identify age-related changes in human meibomian glands that may be associated with meibomian gland dysfunction (MGD). METHODS Excess eyelid tissue from 36 patients (age range, 18-95 years, 19 female, 17 male) who underwent canthoplasty procedures were used. Dermatologic history, age, and presence of MGD were recorded. Samples were frozen, sectioned, and stained with specific antibodies against peroxisome proliferator-activated receptor γ (PPARγ) to identify meibocyte differentiation, Ki67 nuclear antigen to identify cycling cells, and CD45 to identify inflammatory cell infiltration. RESULTS Staining for PPARγ showed cytoplasmic and nuclear localization in the 2 youngest subjects (ages, 18 and 44 years). Older individuals (>60 years) showed predominantly nuclear staining, with cytoplasmic staining limited to the basal acinar cells in 17 of 31 subjects. The number of Ki67 positively stained basal cells were significantly elevated in the younger compared with older subjects based on linear regression analysis (r(2) = 0.35; P < .001). There was also a significant correlation between MG expression grade and CD45 cell infiltration (r = 0.414; P = .05). CONCLUSIONS These results indicate that aging human meibomian glands show decreased meibocyte differentiation and cell cycling that is associated with the development of MGD. Findings also suggest that altered PPARγ signaling may lead to acinar atrophy and development of an age-related hyposecretory MGD. CLINICAL RELEVANCE Meibomian gland dysfunction and evaporative dry eye are common age-related eyelid disorders. Understanding the underlying mechanism of MGD may lead to the development of novel therapeutic strategies to treat this disease.

Age-related changes in the meibomian gland.

The purpose of this study was to characterize the age-related changes of the mouse meibomian gland. Eyelids from adult C57Bl/6 mice at 2, 6, 12 and 24 months of age were stained with specific antibodies against peroxisome proliferator activated receptor gamma (PPARgamma) to identify differentiating meibocytes, Oil Red O (ORO) to identify lipid, Ki67 nuclear antigen to identify cycling cells, B-lymphocyte-induced maturation protein-1 (Blimp1) to identify potential stem cells and CD45 to identify immune cells. Meibomian glands from younger mice (2 and 6 months) showed cytoplasmic and perinuclear staining with anti-PPARgamma antibodies with abundant ORO staining of small, intracellular lipid droplets. Meibomian glands from older mice (12 and 24 months) showed only nuclear PPARgamma localization with less ORO staining and significantly reduced acinar tissue (p < 0.04). Acini of older mice also showed significantly reduced (p < 0.004) numbers of Ki67 stained nuclei. While Blimp1 appeared to diffusely stain the superficial ductal epithelium, isolated cells were occasionally stained within the meibomian gland duct and acini of older mice that also stained with CD45 antibodies, suggesting the presence of infiltrating plasmacytoid cells. These findings suggest that there is altered PPARgamma receptor signaling in older mice that may underlie changes in cell cycle entry/proliferation, lipid synthesis and gland atrophy during aging. These results are consistent with the hypothesis that mouse meibomian glands undergo age-related changes similar to those identified in humans and may be used as a model for age-related meibomian gland dysfunction.

Reducing peak corneal haze after photorefractive keratectomy in rabbits: Prednisolone acetate 1.00% versus cyclosporine A 0.05%

PURPOSE: To compare the effects of topical cyclosporine A 0.05% (Restasis) with those of prednisolone acetate 1.00% (Pred Forte) on corneal haze after photorefractive keratectomy (PRK). SETTING: Gavin Herbert Eye Institute, University of California, Irvine–Orange, California, USA. DESIGN: Experimental study. METHODS: After −9.00 diopter PRK, 15 rabbits were divided into 3 groups and treated for 4 weeks with prednisolone acetate 1.00% or cyclosporine A 0.05% or neither (control). Corneal haze was measured by in vivo confocal microscopy preoperatively and 2, 4, 6, 8, and 12 weeks postoperatively. At 12 weeks, the corneas were evaluated for collagen organization by ex vivo 2‐photon second‐harmonic generation and stromal cell density. RESULTS: Corneal haze was significantly less in the prednisolone acetate group than in the cyclosporine and control groups during the first 6 weeks postoperatively (P<.02). At 8 weeks, there was no significant difference between the 3 groups. There was no significant difference in haze between the cyclosporine and control groups at any time. The stroma was also significantly thinner in the prednisolone acetate group than in the other groups for the first 4 weeks postoperatively (P<.02). Second‐harmonic generation scar thickness measurements at 12 weeks were not significantly different between the groups, although the prednisolone acetate group tended to have lower stromal cell density. CONCLUSION: Cyclosporine A 0.05% had no effect on wound healing after PRK, while prednisolone acetate 1.00% significantly reduced peak corneal haze but had no effect on long‐term corneal haze after discontinuation of the drug. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned.

Volumetric reconstruction of the mouse meibomian gland using high-resolution nonlinear optical imaging.

Recent studies suggest that mouse meibomian glands (MG) undergo age‐related atrophy that mimics changes seen in age‐related human MG dysfunction (MGD). To better understand the structural/functional changes that occur during aging, this study developed an imaging approach to generate quantifiable volumetric reconstructions of the mouse MG and measure total gland, cell, and lipid volume. Mouse eyelids were fixed in 4% paraformaldehyde, embedded in LR White resin and serially sectioned. Sections were then scanned using a 20× objective and a series of tiled images (1.35 × 1.35 × 0.5 mm) with a pixel size of 0.44 μm lateral and 2 μm axial were collected using a Zeiss 510 Meta LSM and a femtosecond laser to simultaneously detect second harmonic generated (SHG) and two‐photon excited fluorescence (TPEF) signals from the tissue sections. The SHG signal from collagen was used to outline and generate an MG mask to create surface renderings of the total gland and extract relevant MG TPEF signals that were later separated into the cellular and lipid compartments. Using this technique, three‐dimensional reconstructions of the mouse MG were obtained and the total, cell, and lipid volume of the MG measured. Volumetric reconstructions of mouse MG showed loss of acini in old mice that were not detected by routine histology. Furthermore, older mouse MG had reduced total gland volume that is primarily associated with loss of the lipid volume. These findings suggest that mice MG undergo “dropout” of acini, similar to that which occurs in human age‐related MGD. Anat Rec, 2011.

Effects of hyperthyroidism on the rectus muscles in mice.

Background: Structural details of vertebrate extraocular muscles (EOMs) have shown an anatomically and functionally distinct laminar organization into an outer orbital (OL) and an inner global layer (GL). Since hyperthyroidism alters tissue oxidative metabolism through mitochondrial enzymes, it is expected that structural/mitochondrial changes may be seen in hyperthyroid EOMs. We investigated the alterations in the laminar organization and mitochondrial changes in hyperthyroid mouse EOMs. Methods: Hyperthyroidism was induced in C57BL/6 mice and fresh rectus muscles were obtained to identify functional mitochondria using MitoTracker® Green and confocal microscopy; frozen sections from rectus muscles were stained with anti-rabbit Troponin T (selectively present in the OL) to demonstrate changes in the OL and GL of the EOMs. Ultrastructural features of EOMs were studied using transmission electron microscopy (TEM). Results: Of all four rectus EOMs studied, the maximum change was seen in the inferior rectus muscle (IR) followed by medial rectus (MR). Myofiber cross-sectional area measurements and Troponin T staining in the control IR EOMs demonstrated a smaller OL (113.2 ± 3.66 μm2) and higher density staining with Troponin T (90%) and a larger GL (411 ± 13.84 μm2) with low intensity staining (10%), while hyperthyroidism resulted in an increased OL (205.9 ± 5.3 μm2) and decreased GL (271.7 ± 7.5 μm2) p = 0.001. Confocal microscopy demonstrated an intense staining especially in the outer rims in the hyperthyroid IR which was confirmed by TEM showing structural alterations in the mitochondria and a subsarcolemmal migration. Conclusions: The outer, thinner, OL of the mouse EOM contains smaller diameter myofibers and fewer mitochondria while the inner, larger GL contains larger diameter myofibers and larger density of mitochondria. Hyperthyroidism results in a significant alteration in the laminar organization and mitochondrial alterations of mouse EOMs.